This invention was made with Government support under Grant No. NIDDK 30111 awarded by National Institute of Diabetes, Digestive and Kidney Disorders. The Government has certain rights in the invention.
The present invention relates to the regulation of a gene product. In particular, a gene product is regulated by an inducible, tissue-specific expression vector that produces an antisense RNA sequence to the messenger RNA (mRNA) of the gene product targeted for regulation.
It is well known that a cell manufactures protein by transcribing the DNA of the gene for that protein. In particular, one of the strands of DNA of the gene is transcribed by an enzyme, RNA polymerase, to produce mRNA. The mRNA molecule has a base sequence that is complementary to that of the transcribed DNA strand. The mRNA is then processed by the removal of introns. The base sequence of the mRNA is translated into the amino acid sequence of a protein molecule by means of the genetic code. This translation process requires many enzymes and a set of transfer RNA (tRNA) molecules, which align the amino acids according to the codon sequence. The translation of mRNA into protein occurs on ribosomes. The translated protein is called a gene product.
The normal development and functioning processes of cells and organisms require that the gene products necessary to carry out these processes are available in the appropriate amounts and at the appropriate times. For example, certain gene products must be present in all cells for many fundamental physiological processes to occur. Other gene products have tissue-specificity and are only necessary in certain cells or tissues. Some gene products are continuously present either in all cells or in certain cells or tissues. Still other gene products are required at different times during development.
Gene regulation alters the quantity or quality of a gene product. These alterations can be used to ascertain the molecular activities of the normal gene product counterpart. Additionally, gene regulation can be used to manipulate cells and organisms at the genetic level. Gene regulation provides scientists and physicians with an expanded ability to study and treat disease processes. In particular, gene regulation techniques have proven especially useful in the elucidation and diagnosis of many diseases and abnormalities. It is now possible to use gene regulation for therapeutic intervention and treatment at the genetic level. In addition, gene regulation can be used to identify and characterize genes involved in fundamental embryological or cellular processes. The identification and characterization of these genes had previously been hampered by the fact that mutations in such genes are often lethal or are recessive in diploid organisms. As discussed above, the selective inactivation or regulation of genes has many potential uses.
Certain genes are regulated at the level of transcription. Transcriptional regulation is carried out either negatively (repressors) or positively (activators) by a protein factor. Specific protein factors regulate translation of specific mRNAs. Recently, it has become evident that RNAs are also involved in regulating the expression of specific genes.
Different approaches have attempted to regulate gene expression by selectively inactivating genes. For example, one way to regulate gene expression is to introduce into living cells, drugs or antibodies that are specific inhibitors of translation or transcription. The inhibition of translation has been efficiently carried out by antibiotics and other inhibitors of protein synthesis. Antibody neutralization and pharmacological perturbation, however, require prior preparation and/or characterization of target and inhibitor which can be time consuming. Further, in multicellular organisms it is difficult to regulate gene expression in a tissue-specific manner since many translational inhibitors are administered systemically.
Another approach to gene regulation is gene disruption. Gene disruption is accomplished using recombinant DNA techniques. It is a process of sequential elimination of both of the alleles for a particular gene. The alleles are eliminated by introducing a mutation into the gene, at the single cell stage of the organism, which renders the gene nonfunctional. Gene disruption is technically difficult and labor intensive. Many times additional genes are unintentionally disrupted. In addition, the gene products eliminated by gene disruption are not present in the organism or cell at any stage of development. This technique leads to a lethal outcome if the gene product is a protein that is necessary for development. Therefore, gene disruption cannot be used for the regulation of gene products that are necessary for normal pre-natal development.
Recently it has been discovered that complementary oligonucleotide inhibition of specific mRNAs can be used to inhibit the expression of a gene product. It is believed that complementary oligonucleotide inhibition of specific mRNAs will have many potential uses. For example, it is believed that it can be used to determine the role of a previously identified gene sequence in different tissues or related species. Further, this method could help to elucidate the function or functions of a gene without the prerequisite of identifying, isolating or characterizing the protein product. Moreover, complementary nucleotide sequences could be used to interfere with structures or activities of RNAs that are never translated. Finally, this approach may represent a general scheme for the functional analysis of cloned gene sequences.
Many approaches have attempted to utilize complementary oligonucleotide inhibition of specific mRNAs to regulate the expression of a gene product. For example, a direct biochemical intervention method that inhibits the expression of a gene product uses the microinjection of a complementary oligonucleotide to inhibit a specific mRNA. This method specifically blocks the translation of a gene's mRNA by RNA--RNA hybridization in vivo. The translation block prevents the synthesis of the gene product. In particular, it is possible to block the translation of a specific mRNA in vivo by microinjection of complementary (antisense) RNA. For instance, RNA complementary to globin mRNA was synthesized in vitro by transcription of an inverted globin cDNA clone. After injection into frog oocyte cytoplasm, the antisense globin RNA forms a hybrid with globin mRNA and selectively prevents its translation. This approach is an effective method of gene regulation. However, the injected antisense RNA molecules have a relatively short life-span before they are degraded or otherwise rendered unavailable for binding to the target mRNA. In addition, this is not a practical approach for the regulation of genes in multicellular organisms since the antisense RNA must be injected into individual cells.
Another gene regulation approach to antisense RNA inhibition of specific mRNAs uses DNA constructs directing the transcription of an antisense RNA strand. The RNA transcript has a sequence complementary to a target mRNA. The antisense RNA anneals to the mRNA and disrupts normal processing or translation. The antisense constructs can be introduced into eukaryotic cells by transfection or microinjection, and function in both transient and stable transformation assays. Antisense transcripts complementary to the target gene mRNA specifically suppress gene activity.
Conditional antisense inhibition can be accomplished with the use of hormone-inducible promoter sequences. For example, antisense DNA constructs were prepared by flipping a gene fragment of interest and inserting this sequence between a promoter and a polyadenylation site in inverse orientation. These antisense plasmids were then co-injected, co-transfected or co-transformed with normal sense DNA plasmid for the gene of interest into cells. While the gene of interest was regulated in the presence of the antisense plasmids, this approach is not amenable to adaptation for gene regulation in a multicellular organism.
Even if this approach were feasible in a multicellular organism, the induction of the antisense RNA would affect the production of the gene product in the whole organism, and not only in specific tissues or organs. This lack of tissue-specificity is problematic especially since the gene product can be necessary in some tissues. Additionally, the size of the gene fragment that is inserted into the plasmid in inverse orientation may be rather large. If the gene fragment is large, it is probable that the antisense RNA produced will have homology to more than just the target mRNA. This homology will suppress the expression of other gene products that are not targeted for regulation.
An example of the use of an antisense RNA transcript is the regulation of guanosine triphosphate-binding regulatory proteins (G proteins). G proteins are key elements in transmembrane signaling and have been implicated as regulators of more complex biological processes such as differentiation and development. G proteins propagate signals from cell surface receptors to a diverse group of effectors that includes adenylylcyclase, phospholipase C, and cation channels. Visual transduction, neuronal signaling, cell growth and differentiation, and metabolic pathways such as glycogenolysis and gluconeogenesis are mediated by way of G proteins. The G protein G.alpha..sub.i2 has been implicated in the inhibition of adenylylcyclase and oncogenesis.
G protein-linked responses regulate many cellular processes in vivo such as the hormonal regulation of metabolic pathways such as lipolysis, glycogenolysis, and gluconeogenesis. The G proteins G.sub.s and G.sub.i regulate these pathways by altering the activity of adenylylcyclase and hence the intracellular amounts of cAMP. G.alpha..sub.s and G.alpha..sub.i have also been implicated in oncogenesis and differentiation. Constitutively active mutants of G.alpha..sub.s and G.alpha..sub.i2 subunits have been identified in pituitary, thyroid, ovarian, and adrenal tumors. G.alpha..sub.s and G.alpha..sub.i2 also regulate adipogenesis in mouse 3T3-L1 cells and stem cell differentiation of F9 teratocarcinoma cells into primitive endoderm.
For example, a DNA sequence having 39 bases that transcribe an RNA antisense to 39 bases of G.alpha..sub.i2 may be used to inhibit expression of this important G protein gene. This 39 base sequence is selected for use in preparing an antisense construct to take advantage of the diversity of the nucleotide sequence in this region and to provide specificity. The pLNCX vector, which contains an ampicillin gene (Amp.sup.r) and neomycin resistance (Neo.sup.r) and retroviral packaging genes (.PSI..sup.+) under the control of the mouse Moloney virus long terminal repeats (5' and 3' long terminal repeats), may be used. The antisense sequences are transcribed under the control of the cytomegalovirus promoter. The expression of the G.alpha..sub.i2 antisense RNA is constitutive. The small size of the antisense sequence ensures specificity. This approach overcomes the problems associated with an antisense RNA having homology with more mRNAs than those targeted for regulation. However, since the antisense RNA is continuously produced, it is not possible to use this approach to regulate genes having gene products that are necessary at different developmental stages.
In view of the foregoing, there still exists a need for gene regulation methods that are simultaneously inducible and tissue-specific, and that utilize an antisense DNA construct transcribing an RNA sequence antisense to the mRNA of a gene product targeted for regulation.